PA1-090

antibody from Invitrogen Antibodies

Targeting: SLC9A3R1 EBP50, NHERF, NHERF1

Provider product page for PA1-090

Western blot

Antibody data

Antibody Data
Antigen structure
References [41]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [1]
Immunohistochemistry [1]
Flow cytometry [1]
Other assay [23]

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Product number: PA1-090 - Provider product page
Provider: Invitrogen Antibodies
Product name: EBP50 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Other
Description: PA1-090 detects ezrin-radixin-moesin (ERM) binding phosphoprotein of 50 kDa (EBP50) from mouse tissues, human T84 and Calu3 cells as well as recombinant human protein.
Concentration: 1 mg/mL

SLC9A3R1 protein structure - PA1-090 shown in red.

Supportive validation

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Experimental details: Western blot detection of recombinant human EBP50 using Product # PA1-090.

Supportive validation

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Experimental details: Western blot analysis was performed on whole cell extracts of T-47D (Lane 1), MCF7 (Lane 2), HepG2 (Lane 3) and Caco-2 (Lane 4). The blots were probed with Anti- EBP50 Rabbit polyclonal Antibody (Product # PA1-090, 1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A ~ 50 kDa band corresponding to EBP50 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

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Experimental details: Western blot was performed using Anti-EBP50 Polyclonal Antibody (Product # PA1-090) and a 50 kDa band corresponding to EBP50 was observed across cell lines tested except MDA-MB-231 which is reported to be negative. Membrane enriched extracts (30 µg lysate) of MCF-7 (Lane 1), T-47D (Lane 2), SK-BR-3 (Lane 3), MDA-MB-231 (Lane 4), HeLa (Lane 5) and Hep G2 (Lane 6), were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (0.2µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

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Supportive validation

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Experimental details: Figure 2 Cldn6 induces the formation of cell-cell junctions and apicobasal cell polarity in F9 stem cells. (A and D) X-Y and Z projections of F9:Cldn6 cells stained for Cldn6 together with the tight-junction markers (Cldn7, Ocln, ZO-1, and ZO-1alpha+ variant), the basolateral maker (E-Cad), and the apical markers (ezrin, radixin, and EBP50). The arrowhead indicates the recruitment of Ocln to a part of Cldn6-positive premmature cell-cell junctions. The asterisk shows the absence of EBP50 on the apical surfaces of undifferentiated cells possessing Cldn6-positive premature cell-cell junctions. Scale bars, 20 um. (B) Confocal images of undifferentiated areas of F9:Cldn6 cells stained for Cldn6 together with Ocln. The arrowheads indicate the partial recruitment of Ocln to Cldn6-positive cell borders of undifferentiated cells. Scale bar, 20 um. (C) Confocal images of F9:Cldn6 cells stained for Cldn6 together with Cldn4 and DAPI. Scale bar, 20 um.

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Experimental details: Figure 5. CEACAM1 can be excluded from the CMD by adding tails from NHERF1-binding proteins. (A) GFP-CEACAM1-based constructs differ in their exclusion from the CMD 4 d after seeding. Images were collected with identical microscope settings to Fig. 1 C . (B) Cells were scored for GFP exclusion from the CMD as in Fig. 1 C . Only 5/169 cells appeared to exclude GFP-CEACAM1 from the CMD, and GFP-CEACAM1-DTHL (13/135), GFP-CEACAM1-PODXLDelta4 (10/237), and GFP-CEACAM1-CFTRDelta4 (12/148) were similar, whereas GFP-CEACAM1-PODXL (146/253) and GFP-CEACAM1-CFTR (94/111) were excluded from the CMD in most cells. (C) FRAP measurements indicated that GFP-CEACAM1 constructs that were excluded from the CMD were also less mobile in the apical membrane. (D) There was no significant difference between FRAP measurements of GFP-CEACAM1 and GFP-CEACAM1-DTHL after 120 s; however, there were significant differences between GFP-CEACAM1-PODXL and GFP-CEACAM1-PODXLDelta4, GFP-CEACAM1-CFTR and GFP-CEACAM1-CFTRDelta4, and GFP-CEACAM1-CBP and GFP-CEACAM1-CBPDelta4 (P < 0.005). (C and D) Each FRAP value is a mean of measurements from eight cells. Error bars represent standard deviation between measurements. (E) Cells expressing GFP constructs were grown on tissue culture plates and lysed when confluent. GFP constructs were immunoprecipitated from cell lysates, eluted, and Western blotted. Membranes were probed with anti-NHERF1 and either anti-GFP or anti-gp135 antibodies. The arrow to the left of the po

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Experimental details: Fig. 3 Expression analysis of cytoplasmic NHERF1 in human colorectal cancer. ( A ) Representative images of cytoplasmic NHERF1 immunoreactivity in the primary tumour matched with adjacent cancer-uninvolved colonic mucosa by immunohistochemistry (Original magnification on the left x100, enlargement on the right x200). ( B ) The distribution of cytoplasmic NHERF1(+) cells on normal mucosa and the tumour compartment of the same colonic lesion. (Horizontal bold line = median value; *** P < 0.0001).

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Experimental details: Figure 3 The expression of proteins associated with colorectal carcinoma (CRC) tumorigenesis and metastasis was determined in the novel African American CRC lines by immunoblotting. (A) Qualitative analysis of CHTN06, SB501 and SB521 and HT-29, a Caucasian American CRC cell line, for protein expression of beta-catenin, p53, nuclear factor (NF)-kappaB (p50 and p65), villin-1, MSH2, MSH6, MLH1 and ezrin. (B) Semi-quantitative densitometry was performed by normalizing protein expression to the respective beta-tubulin loading control. Data were generated from three independent experiments. CEA, carcinoembryonic antigen.

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Experimental details: Figure 3. CMD exclusion of GFP-PODXL is dependent on NHERF1. (A) NHERF1 was knocked down in GFP-PODXL cells (NHERF1 shRNA) using a retroviral shRNA system. Control cells expressing GFP-PODXL (vector) were retrovirally transduced with a puromycin resistance plasmid without an shRNA sequence. Cell lysates were blotted with anti-NHERF1 and antiezrin antibodies. Molecular mass is indicated in kilodaltons. (B) Live imaging of cells 4 d after seeding revealed that control cells (vector) exclude GFP-PODXL from the CMD but NHERF1 shRNA cells do not. Exclusion was quantified as in Fig. 1 C , and the percentage of cells excluding GFP-PODXL from the CMD appears on each image. 148/230 control cells (vector) excluded GFP-PODXL from the CMD, whereas very few cells expressing NHERF1 shRNA (8/186) excluded GFP-PODXL. Images were collected with identical microscope settings to Fig. 1 C . (C) Knocking down NHERF1 allowed GFP-PODXL to enter primary cilia. Cells were imaged live 12 d after seeding on filters. (top row) Z stacks of the apical membrane of control (vector) or NHERF1 shRNA live cells expressing GFP-PODXL were projected into a single image. Images were collected with identical microscope settings to Fig. 1 C , but at lower digital zoom. (bottom row) Single confocal sections above the apical membrane were taken from the z stacks in the top row, and image brightness was increased. Visible cilia are labeled with arrowheads. (D) 12 d after seeding, NHERF1 shRNA and control cells (vector)

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Experimental details: Figure 6. CEACAM1 can be excluded from the CMD independently of NHERF1 by adding domains that bind more directly to the cytoskeleton. (A) Addition of NHERF1's ERM-binding domain or ezrin's actin-binding domain is sufficient to exclude GFP-CEACAM1 from the CMD 4 d after seeding. NHERF1 was knocked down in GFP-CEACAM1-NHERF1 cells (GFP-CEACAM1-NHERF1 shRNA), and exclusion was similar to control cells (GFP-CEACAM1-NHERF1 vector). Images were collected with identical microscope settings to Fig. 1 C . (B) GFP-CEACAM1-NHERF1 shRNA and GFP-CEACAM1-NHERF1 vector cell lysates were blotted with anti-NHERF1 and antiezrin antibodies. Molecular mass is indicated in kilodaltons. (C) Cells were scored for GFP exclusion from the CMD as in Fig. 1 C . 113/199 cells expressing GFP-CEACAM1-NHERF1 appeared to exclude the construct from the CMD, and similar results were seen for GFP-CEACAM1-ezrin (105/185). After shRNA knockdown of NHERF1, GFP-CEACAM1-NHERF1 was excluded from the CMD in 193/393 cells, a similar ratio to cells only treated with vector (125/232). Error bars represent standard deviation between experiments. (D) FRAP measurements indicated that GFP-CEACAM1 was less mobile in the apical membrane after addition of an ERM- or actin-binding domain, and this was independent of NHERF1 knockdown. (E) There are significant differences in the FRAP measurements between GFP-CEACAM1 and both GFP-CEACAM1-NHERF1 (**, P < 0.005) and GFP-CEACAM1-ezrin (*, P < 0.05) after 120 s. No significa

Supportive validation

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Experimental details: Fig. 4 Analysis of PAR-2 and cytoplasmic NHERF1 in human colorectal cancer. As shown in ( A ), the correlation between protein expression of PAR-2 and cytoplasmic NHERF1 was evaluated by Spearman's rank correlation coefficient analysis, and a positive significant correlation was established. ( B ) A representative tissue sample stained with PAR-2 and EBP-50 antibodies and detected with Alexa Fluor 568 (red) and Alexa Fluor 488 (green) secondary antibodies, respectively, prior to fluorescence microscopy analysis. Overlaps between red and green signals (merged) point to co-localizations (in yellow). Arrowheads indicate invasive cells disseminated into the stroma close to a blood vessel with a high global expression of two proteins, where PAR-2 co-localized with NHERF1 on cytoplasmic and membranous compartments (scale bar = 20 mum).

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Experimental details: Figure 2 Intestinal defects in NHERF1 -/- mice. (A) IF with NHERF1 antibody and DAPI for highlighting nuclei in NHERF1 -/- and NHERF1 +/+ mice shows high NHERF1 apical PM expression in intestinal epithelium (yellow arrowhead) and in the mesothelial lining of the serosa (arrow). (B) BrdU incorporation in 6-week-old NHERF1 +/+ and NHERF1 -/- female littermates showing similar numbers of BrdU+ cells in both genotypes and upper displacement of the BrdU+ progenitor cells in NHERF1 -/- crypts (right graph). Data are means +- SEM from 50 crypts. The cell position 0 was assigned for the bottom-most cell of every counted crypt. In contrast, NHERF1 -/- spleen BrdU+ counts show significantly enhanced proliferation. (C) H&E, lysozyme IHC for Paneth cells, and Alcian blue special stain for goblet cells of ileum sections show higher numbers of Paneth (arrows in H&E) and goblet cells in NHERF1 -/- versus NHERF1 +/+ mice. (D) Transmission electron microscopy (TEM) shows blunted microvilli in NHERF1 -/- colon epithelium.

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Experimental details: Figure 1 Representative images of the immunoreactivity of beta-catenin, NHERF1 and RASSF1A Membrane and cytoplasmic localization of beta-catenin in ANT A. , membrane, cytoplasmic and nuclear expression in T B. , membrane, cytoplasmic and nuclear beta-catenin staining in LM C. NHERF1 immunoreactivity is present at the apical membrane, in cytoplasm and nucleus in ANT D. , while in T E. and LM F. it becomes mostly cytoplasmic and nuclear. Heterogeneous cytoplasmic staining intensity of RASSF1A in ANT G. , granular cytoplasmic staining in T H., cytoplasmic and nuclear immunoreactivity in LM I. (original magnification x200).

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Experimental details: Figure 1 Immunoreactivity and localization of NHERF1 in breast carcinoma. Representative images of immunohistochemical staining: ( a ) positive staining for cytoplasmic NHERF1 in tissue of poorly differentiated IDC (original magnification on the left x 20) and panoramic view of the tumor (original magnification on the right x 5); ( b ) NHERF1 antibody stained intensely in the cytoplasm and in the nucleus of the cells of poorly differentiated IDC (original magnification on the left x 20), and panoramic view of the tumor (original magnification on the right x 5); ( c ) apical membranous immunoreactivity of NHERF1 in non neoplastic epithelia cells (original magnification x 20)

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Experimental details: Fig. 1 Cryba1 cKO RPE show age-related microvilli defects. a Immunostaining for EBP50 (red) and F-actin (phalloidin, green) on retina sections showed disorganized microvilli in 1-month-old cKO RPE cells, and microvilli loss (arrows) in RPE cells of 9-month-old cKO mice compared to age-matched control. Mag: magnified area outlined in merged image. DAPI (blue). Scale bar: 20 mum. Graph shows the average microvilli height in control and cKO retina sections measured by the length measurement tool in ZEN software based on the staining results. For each biological repeat, three representative values from different RPE locations (center, middle, peripheral) were averaged as the microvillus height. Differential interference contrast (DIC) microscopy showed the area of each sample for imaging. b Z-stack imaging for EBP50 (red) and F-actin (phalloidin, green) with an orthogonal projection showing upright and abundant microvilli in 1-month-old control (arrowhead), but not in cKO RPE cells (arrow). In 4-month-old mice, some cKO RPE cells presented collapsed microvilli indicated by sinking F-actin and EBP50 in the z -direction (arrows). Some RPE cells of 9-month-old cKO mice (asterisks) lacked microvilli completely. Scale bar: 20 mum. c TEM images of cKO and control retina sections. Two-month-old control mice exhibited microvilli very well interdigitated with photoreceptor outer segments (asterisks). Arrows show disorganized microvilli of RPE cells in 2-month-old cKO and 20-month-old cont

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Experimental details: Fig. 2 Disrupted apical-basal polarity in RPE cells lacking Cryba1 . a Western blot analysis of 3-week and 9-month-old RPE extracts showing that ezrin phosphorylation and Rac1 expression were reduced in cKO RPE cells at both ages. b Western blot analysis of 4-month-old RPE extracts confirmed downregulation of EBP50 protein in cKO RPE cells. c Immunostaining for Na + /K + ATPase and MCT3 on sections of 9-month-old retina showed reduced Na + /K + ATPase and mis-located MCT3 in Cryba1 cKO RPE cells (indicated by arrows). Scale bar: 20 mum. d Western blot analysis of RPE extracts from control and cKO mice at different ages showed reduced Na + /K + ATPase; however, there was no significant change in MCT3 abundance in cKO RPE. Statistical analysis was performed using either a two-tailed unpaired Student's t -test ( a , b ) or two-way ANOVA ( d ). * P < 0.05, *** P < 0.001, n = 4~6.

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Experimental details: 2 Correlation of ERM-binding phosphoprotein 50 (EBP50) immunoreactivity and mRNA expression in breast carcinoma. Examples of EBP50 protein and mRNA expression in oestrogen receptor (ER)-positive (IBC1 and IBC2) and ER- (IBC3) breast carcinomas. IBC1, with tumour involved in lymph nodes (N+); IBC2 and IBC3, without tumour involved in lymph nodes (N-). Staining in A , B and C represents immunohistochemical detection of ER protein. Strong ( D ) and weak ( E ) EBP50 immunoreactivity is found in IBC1 and IBC2, respectively, whereas negative staining ( F ) is indicated in IBC3. Similar patterns of immunoreactivity with EBP50 antisense probe are seen in G (strong), H (weak) and I (negative), suggesting that the mRNA expression of EBP50 correlates with its immunoreactivity in these tissue specimens. An EBP50 sense probe was used as a negative control for every specimen by hybridization on a consecutive section (no signals in J , K and L ).

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Experimental details: 4 Independent validation of immunohistochemical study on tissue microarrays. Examples of ERM-binding phosphoprotein 50 (EBP50) protein expression in oestrogen receptor (ER)-positive ( A,B ) and ER- ( C ) breast carcinoma. Strong and weak EBP50 immunoreactivity is found in cases A and B , respectively, whereas negative staining is indicated in case C .

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Experimental details: 2 Na + /H + exchanger regulatory factor 1 (NHERF1) immunolocalization in well-organized and invaded lobules of the mammary gland and lymph node. In the non-tumour compartment ( A ) of the tissue, NHERF1 localized only apically in the epithelial cells lining the lobules, and some lymphocytes that are are not stained with NHERF1 are dispersed in the stroma (arrow). The invaded tissue ( B ) shows NHERF1 immunolocalization in the metastatic spreading cells and in the lymphocytes located in adjacent regions. Lymphocytes in metastatic lymph node ( C ) and peritumoral blood vessels ( D ) also show intense immunoreactivity. Bar: 32 mum.